1. Field of the Invention
This invention relates to a novel alkaline pullulanase Y having an .alpha.-amylase activity, a microorganism producing said alkaline pullulanase Y, and a process for producing said alkaline pullulanase Y.
2. Description of the Background Art
Pullulanase is an enzyme which breaks only .alpha.-1,6-glycosidic linkage of pullulan and finally produces maltotriose. Pullulanase was first discovered from a strain belonging to Aerobacter aerogenes by Bender and Wallenfels in 1961 [Biochem. Z., 334, 79, (1961)]. Recently, various microorganisms capable of producing pullulanase have been reported. These microorganisms are, for example, Bacillus sp. [J. Jpn. Soc. Starch Sci., 30, 200, (1983)]; Bacillus acidopullulyticus [Agric. Biol. Chem. 52, 2293, (1984)]; Bacillus stearothermophilus [Eur. J. Appl. Microbiol. Biotechnol., 17 24 (1983); Streptococcus mitis [Biochem. J., 108, 33, (1968)]; Lactobacillus [Denpun Kagaku, 28, 72, (1981)]; Clostridium sp. [Appl. Environ. Microb., 53, 7, (1987)]; Clostridium thermohydrosulfuricum [Appl. Environ. Microb., 49, 5, (1985), J. Bacteriol., 164, 3, (1985), Biochem. J., 246, (1987)], Thermus aquaticus [Enzyme Microb. Technol., 8, (1986]); and Thermus sp. [J. Jpn. Soc. Starch Sci., 34, 1 (1987)].
It is known that pullulanase not only possesses activities against pullulan, but also hydrolysis activities against .alpha.-1,6-glycosidic linkage of starch, glycogen, amylopectin, as well as against branched oligosaccharides produced by their partial decomposition. Because of this characteristic, pullulanase is called a "debranching enzyme".
Further, it has been found that pullulanase used in combination with both endo-type amylase and exo-type amylase could yield glucose or maltooligo-saccharides such as maltose, maltotriose, maltotetraose, maltopentaose, or maltohexaose from starch in a high yield. This characteristic has lately attracted considerable attention.
In addition, development of pullulanase having .alpha.-amylase activity capable of reacting with an .alpha.-1,4-glycosidic linkage has been desired in order to simplify the conventional sugar-manufacturing processes in which two or more of enzymes are being used. In this respect, only two enzymes have been reported. One is a combined enzyme of pullulanase-amylase produced by Bacillus subtilis TU (Agric. Biol. Chem., 51, 9, (1987); Japanese Patent Publication No. 18717/1989) and the other is an amylase having pullulanase activity produced by Bacillus circulans (Japanese Patent Laid-open No. 60376/1989).
Meanwhile, it has been elucidated that the combined use of a pullulanase having the above-mentioned characteristics together with an .alpha.-amylase as additives for dish-washing detergents or laundry detergents remarkably improves detergency against mainly starch soil (Japanese Patent Application No. 285424/1988). Further utilization of pullulanase is expected in these fields.
However, almost all naturally occuring pullulanases are classified into neutral or acidic pullulanases which exhibit the maximum and stable enzymatic activity in neutral or acidic conditions. There exists few alkaline or alkali-resistant pullulanases having a better stability and exhibiting the maximum activity at an alkaline pH range at which cloth-washing or dish-washing is performed. As for an alkaline pullulanase having .alpha.-amylase activity, such a kind of alkaline pullulanase has never been discovered until now. An alkaline pullulanase herein denotes that having an optimum pH at an alkaline range. An alkali-resistant pullulanase denotes that having an optimum pH in a neutral to acidic range, but having a sufficient degree of activities in an alkaline range as it has at its optimum pH while retaining a good stability. The word "neutral" herein are defined as a pH range of 6-8 and the word "alkaline" designates a pH range of not less than 8.
No processes for producing alkaline pullulanase nor alkali-resistant pullulanase is heretofore known, except for a report of Horikoshi et al., which discloses a process for producing alkali pullulanase through culturing an alkalophilic microorganism belonging to genus Bacillus [Biochem, Biophys. Acta, 397, 188, (1975), and Japanese Patent Publication No. 27786/1978).
Pullulanase of Horikoshi et al. is an enzyme having its optimum pH at an alkaline range and possessing wider substrate specificity than conventionally known pullulanases. However, since its optimum pH is in a weak alkaline range of 8-9 and lacks in .alpha.-amylase activity, it is not applicable to detergent compositions. In addition, the pullulanase of Horikoshi et al. has a disadvantage that the enzyme is unstable and the productivity of enzyme is low. This type of process is therefore unsuitable for industrial fermentative production. Because of these reasons, the development of pullulanases having an optimum pH in higher alkaline range and having .alpha.-amylase activity as well has been desired.
Dish-washing or cloth-washing is usually carried out in a wide pH range of from neutral to high alkaline range. It is therefore worthwhile to discover and obtain natural microorganisms having an optimum pH at highly alkaline range and capable of producing alkaline pullulanase having .alpha.-amylase activity which functions as an enzyme usable for dish-washing and cloth-washing detergents.
In view of this situation, the present inventors have carried out extensive studies in order to obtain natural microorganisms capable of producing alkaline pullulanase having .alpha.-amylase activity, and, as a result, found that an alkalophilic microorganism Bacillus sp. KSM-AP1378 (FERM P-10886) which was discovered by the inventors in the soils in Tochigi-shi, Tochigi-ken, Japan, was capable of producing a novel alkaline pullulanase Y having .alpha.-amylase activity which was effective as a detergent component for automatic dishwasher or for a laundry detergents. This finding has led to the completion of the present invention.